Preparation of DNA Containing 5-Hydroxymethyl-2′-Deoxycytidine Modification Through Phosphoramidites with TBDMS as 5-Hydroxymethyl Protecting Group

Abstract

This unit describes procedures for preparation of two phosphoramidite building blocks III and IV, both containing a TBDMS as 5-CH₂OH-protecting group. Phosphoramidites III and IV allow efficient incorporation of 5-hmC into DNA and a “one-step” deprotection procedure to cleanly remove all the protecting groups. A “two-step” deprotection strategy is compatible with ultramild DNA synthesis, which enables the synthesis of 5hmC-containing DNA with additional modifications. Methods are also presented for their incorporation into oligonucleotides by solid-phase synthesis, subsequent deprotection, and HPLC analysis.

INTRODUCTION

The discovery of 5-hydroxymethylcytosine (5-hmC) in the mammalian genome of certain cell types as the “sixth” base in 2009 (Kriaucionis and Heintz, 2009; Tahiliani et al., 2009) has triggered tremendous studies in this area. A group of Tet dioxygenases have been shown to utilize dioxygen to oxidize 5-mC to 5-hmC in the mammalian genome, and display important functions in the maintenance and normal myelopoiesis of embryonic stem cells (ES cells; Tahiliani et al., 2009; Ito et al., 2010; Ko et al., 2010). All results so far have suggested that 5-hmC is an important epigenetic modification (Loenarz and Schofield, 2009). In order to facilitate the biological study of 5-hmC in DNA, an efficient synthesis of 5-hmC-containing DNA as a model substrate is a prerequisite. In addition, DNA with other labeling, such as fluorophores or biotin, is usually needed for biochemical characterizations, whose incorporation into DNA often requires or prefers the use of ultramild DNA synthesis. Therefore, it is also highly desirable to develop 5-hmC phosphoramidite building blocks, which are compatible with ultramild DNA synthesis.

Before we started this work, two phosphoramidite building blocks (I and II, Fig. 4.47.1; Tardy-Planechaud et al., 1997; De Kort et al., 2001; Hansen et al., 2011) have been developed for the incorporation of 5-hmC into DNA (Fig. 4.47.1). These methods are currently used in the synthesis of 5-hmC-containing DNAs for biological studies. However, both methods pose limitations in the post-synthetic removal of the protecting groups.

Figure 4.47.1.

Two reported phosphoramidite building blocks of 5-hydroxymethyl-2′-deoxycytidine.

Phosphoramidite I uses Ac as the protecting group for the 5-hydroxymethyl (5-CH₂OH) group. After its incorporation into DNA, the post-synthetic treatment with NH₄OH generates an amide byproduct, which is produced by the S_N2 reaction of ammonia attacking the pseudobenzylic carbon with the OAc group as the leaving group to give an amine intermediate followed by migration of the benzoyl group (De Kort et al., 2001). To avoid the formation of this byproduct, strong base treatment (0.1 M NaOH in dioxane/H₂O) is required. Phosphoramidite II with a cyanoethyl as the protecting group of 5-CH₂OH is more widely used since it is commercially available. However, the post-synthetic removal of the cyanoethyl group turned out to be troublesome (Tardy-Planechaud et al., 1997). Treatment of synthetic DNA with NH₄OH at 65°C for 60 hr could not completely remove the protecting group. Additional treatment with stronger base (NaOMe in MeOH) is necessary. Obviously, neither of the phosphoramidites reported is suitable for synthesizing 5-hmC-containing DNA with additional base-labile modifications. Therefore, it is highly desirable to develop new 5-hmC phosphoramidites using protecting groups that can be cleanly removed under mild conditions. Herein we report the efficient syntheses of two 5-hmC phosphoramidite building blocks (III and IV), and demonstrate the advantages in the removal of the protecting groups following oligo synthesis.

We chose TBDMS as the 5-CH₂OH-protecting group for phosphoramidites III and IV for two reasons: (1) unlike OAc at the pseudobenzylic position, O-TBDMS is not a good leaving group and thus will not result in the formation of the S_N2 byproduct during NH₄OH treatment; (2) TBDMS may be readily removed using fluoride treatment such as ammonium fluoride.

CAUTION: Carry out all operations involving organic solvents and reagents in a well-ventilated fume hood, and wear protective glasses and gloves.

NOTE: Evaporation of solvents is carried out under reduced pressure at temperatures below 35°C.

PREPARATION OF KEY INTERMEDIATE 3′,5′-O-di-t-BUTYLSILYL-5-t-BUTYLDIMETHYLSILOXYMETHYL-2′-DEOXYURIDINE (S.5)

To synthesize phosphoramidites III and IV, intermediate S.5 is the key intermediate. We have tried several methods and found the route depicted in Figure 4.47.2 is the most efficient (Dai et al., 2011). Crouch et al. reported that 5-iodo-3′,5′-di-O-TBDMS-2′-deoxyuridine could be converted to the corresponding 5-formyl-dU analogue in 82% yield using standard Stille coupling conditions (Crouch and Eaton, 1994). We reasoned that the reduction of the aldehyde analogue should generate the corresponding alcohol. Our new synthesis started from a commercial reagent, 5-iodo-2′-deoxyuridine (S.1, Fig. 4.47.2). We chose to protect the 3′ and 5′-hydroxyls with a di-t-butylsilyl group so that it could be selectively removed after 5-CH₂OH is protected by TBDMS. Thus, S.1 was converted into S.2 in 92% yield. The Stille reaction generated the corresponding 5-formyl-dU analogue S.3 in 85% yield. Reduction of S.3 with NaBH₄ in the presence of CeCl₃ gave the corresponding 5-hmdC analogue S.4 in 78% yield. Protection of the hydroxyl group with TBDMS provided S.5 in 95% yield. The product for each step could be easily purified using low-polarity eluents. The overall yield of S.5 reaches to 58% from S.1.

Figure 4.47.2.

Synthesis of key intermediate S.5.

Materials

• 5-Iodo-2′-deoxyuridine (Berry Associate)

• N,N-Dimethylformamide (DMF, anhydrous, 99.8%, Sigma-Aldrich)

• Argon

• Di-tert-butylsilyl-bis(trifluoromethanesulfonate) (97%, Sigma-Aldrich)

• Imidazole (>99%, Sigma-Aldrich)

• Ethyl acetate (EtOAc; >99%, Fisher)

• 5% NaHCO₃ solution (see recipe)

• Brine (see recipe)

• Na₂SO₄

• Silica gel (Sigma-Aldrich)

• Methylene chloride (CH₂Cl₂; HPLC grade, Fisher)

• Methanol (MeOH; HPLC grade, Fisher)

• Toluene (99.8%, anhydrous, Sigma-Aldrich)

• Triphenylphosphine (Ph₃P; 99%, Sigma-Aldrich)

• Pd₂(dba)₃ (97%, Sigma-Aldrich)

• Carbon monoxide (Airgas)

• Tributyltin hydride (Bu₃SnH; 97%, Sigma-Aldrich)

• Methanol (MeOH; anhydrous, 99.8%, Sigma-Aldrich)

• Cerium(III) chloride heptahydrate (99.9%, Sigma-Aldrich)

• Sodium borohydride (NaBH₄; >99.9%, Sigma-Aldrich)

• t-Butyldimethylsilyl chloride (TBDMS-Cl; 97%, Sigma-Aldrich)

• 100-mL round-bottom flasks

• Ice bath

• Rotary evaporator connected to vacuum pump (delivering ~100 micro max vacuum), chilled by dry-ice

• 2 × 40–cm glass columns

• Self-contained glass coupling apparatus equipped with a pressure-equalizing addition funnel (Chemglass)

• Silicone oil bath (Fisher)

• Additional reagents and equipment for thin-layer chromatography (APPENDIX 3D)

Prepare 3′,5′-O-di-t-butylsilyl-2′-deoxyuridine (S.2)

• 1Place 2.05 g (4.06 mmol) of 5-iodo-2′-deoxyuridine (S.1) in a 100-mL round-bottom flask.
• 2Add 20 mL anhydrous DMF under argon to give a cloudy solution. Cool the mixture to 0°C using an ice bath.
• 3Add 2.39mLdi-tert-butylsilyl-bis(trifluoromethanesulfonate) (1.1 eq.) dropwise (the cloudy solution turns clear). Keep the mixture stirring at 0°C for 10 min.
• 4Remove the ice bath and allow the temperature of the reaction to reach room temperature.
• 5Add 0.98 g imidazole (2.5 eq.) and stir the mixture at room temperature for 0.5 hr.
• 6Evaporate DMF using a rotary evaporator connected to a vacuum pump.
• 7Add 100 mL EtOAc to the residue and wash the solution with 50 mL deionized water, 50 mL of 5% NaHCO₃ solution, and 50 mL brine, dry over Na₂SO₄, and filter by gravity filtration (e.g., place a piece of Whatman filter paper on a conical funnel).
• 8Evaporate the filtrate to dryness using a rotary evaporator to afford a crude residue.
• 9Pack a 2 × 40–cm glass column 20-cm high with silica gel in CH₂Cl₂. Apply the crude residue dissolved in a minimum of CH₂Cl₂. Elute the desired product using 0% to 3% (v/v) HPLC-grade MeOH in CH₂Cl₂.
• 10Monitor fractions by TLC (APPENDIX 3D) using 3% HPLC-grade MeOH in CH₂Cl₂ and combine fractions containing product. Remove solvents and dry the resulting residue using a high-performance vacuum pump to afford analytically pure nucleoside S.2 as white foam.
• 11
  Characterize the product, S.2, by ¹H NMR, ¹³C NMR, and HRMS.

  3′,5′-O-Di-t-butylsilyl-5-iodo-2′-deoxyuridine (S.2) Yield 2.63 g (92%); ¹H NMR (500 MHz) (CDCl₃) δ: 9.86 (br., 1H), 7.66 (s, 1H), 6.13 (m, 1H), 4.46 (m, 1H), 4.20 (m, 1H), 4.01 (m, 1H), 3.71 (m, 1H), 2.41 (m, 2H), 1.08 (s, 9H), 0.99 (s, 9H). ¹³C NMR (125.8 MHz) (CDCl₃) δ: 160.2, 150.0, 144.2, 84.8, 78.3, 74.6, 69.0, 67.3, 39.0, 27.5, 27.2, 22.8, 20.2. HRMS calculated for C₁₇H₂₈IN₂O₅Si, [MH⁺] 495.0812 (calcd.), 495.0807 (found).

Prepare 3′,5′-O-di-t-butylsilyl-5-formyl-2′-deoxyuridine (S.3)

• 12Place 2.01 g 3′,5′-O-di-t-butylsilyl-5-iodo-2′-deoxyuridine (5.79 mmol, S.2) in a 100-mL round-bottom flask with a self-contained glass coupling apparatus equipped with a pressure-equalizing addition funnel.
• 13Add 50 mL anhydrous toluene, 0.64 g Ph₃P (0.6 eq.), and 0.42 g Pd₂(dba)₃ (0.40 mmol, 0.10 eq.).
• 14Charge the apparatus with 50 psi of carbon monoxide (CO) and heat to 70°C in an oil bath.
• 15Add 1.19 mL Bu₃SnH (1.05 eq.) slowly with a syringe within 1 hr. After that, keep stirring the mixture for 2 hr at 70°C and then cool to room temperature.
• 16Filter to remove the small amount of solid.
• 17Evaporate the filtrate to dryness using a rotary evaporator to afford a crude residue.
• 18Pack a 2 × 40–cm glass column 20-cm high with silica gel in CH₂Cl₂. Apply the crude residue dissolved in a minimum of CH₂Cl₂. Elute the desired product using 0% to 3% (v/v) MeOH in CH₂Cl₂.
• 19Monitor fractions by TLC (APPENDIX 3D) using 3% MeOH in CH₂Cl₂ and combine fractions containing product. Remove solvents and dry the resulting residue using a high-performance vacuum pump to afford analytically pure nucleoside S.3 as white foam.
• 20
  Characterize the product, S.3, by ¹H NMR, ¹³C NMR, and HRMS.

  3′,5′-O-Di-t-butylsilyl-5-formyl-2′-deoxyuridine (S.3) Yield 1.37g (85%); ¹H NMR (500 MHz) (CDCl₃) δ: 10.03 (s, 1H), 8.60 (br., 1H), 8.21 (s, 1H), 6.13 (m, 1H), 4.49 (m, 1H), 4.17 (m, 1H), 4.08 (m, 1H), 3.77 (m, 1H), 2.45 (m, 2H), 1.07 (s, 9H), 1.04 (s, 9H). ¹³C NMR (125.8 MHz) (CDCl₃) δ: 186.7, 162.1, 149.7, 145.3, 112.1, 86.5, 79.6, 75.1, 68.1, 40.1, 28.4, 28.1, 23.6, 21.1. HRMS calculated for C₁₈H₂₉N₂O₆Si, [MH⁺] 397.1795, (calcd.), 397.1789 (found).

Prepare 3′,5′-O-di-t-butylsilyl-5-hydroxymethyl-2′-deoxyuridine (S.4)

• 21Place 1.50 g 3′,5′-O-di-t-butylsilyl-5-formyl-2′-deoxyuridine (3.79 mmol, S.3) in a 100-mL round-bottom flask, add 40 mL anhydrous MeOH and 4.24 g CeCl₃.7H₂O (3.0 eq.) under argon.
• 22Cool the mixture to 0°C using an ice bath and add 144 mg NaBH₄ (1.0 eq.) slowly (within 15 min).
• 23
  Remove the ice bath and stir the reaction mixture at room temperature for 30 min.

  TLC analysis shows that S.8 is completely consumed with a slower new spot formed.

• 24Add 5.0 g silica gel and remove the volatile under reduced pressure to give a residue.
• 25Pack a 2 × 40–cm glass column 20-cm high with silica gel in CH₂Cl₂. Apply the crude residue on the top of the column. Elute the desired product using 0% to 4% MeOH in CH₂Cl₂.
• 26Monitor fractions by TLC (APPENDIX 3D) using 4% MeOH in CH₂Cl₂ and combine fractions containing product. Remove solvents using a rotary evaporator and dry the resulting residue using a high-performance vacuum pump to afford analytically pure nucleoside S.4 as white foam.
• 27
  Characterize the product, S.4, by ¹H NMR, ¹³C NMR, and HRMS.

  3′,5′-O-Di-t-butylsilyl-5-hydroxymethyl-2′-deoxyuridine (S.4) Yield 1.18 g (78%). ¹H NMR (500 MHz) (CDCl₃) δ: 10.10 (br., 1H), 7.34 (s, 1H), 6.20 (t, J=5.0 Hz, 1H), 4.34–4.45 (m, 3H), 4.21 (m, 1H), 4.01 (m, 1H), 3.68 (m, 1H), 2.36 (m, 2H), 1.06 (s, 9H), 1.00 (s, 9H). ¹³C NMR (125.8 MHz) (CDCl₃) δ: 164.7, 151.1, 138.1, 115.3, 85.0, 79.0, 75.7, 68.2, 59.2, 39.6, 28.4, 28.1, 23.6, 21.1. HRMS calculated for C₁₈H₃₁N₂O₆Si, [MH⁺] 399.1951 (calcd.), 399.1946 (found).

Prepare 3′,5′-O-di-t-butylsilyl-t-butyldimethylsiloxymethyl-2′-deoxyuridine (S.5)

• 28Place 1.00 g 3′,5′-O-di-t-butylsilyl-5-hydroxymethyl-2′-deoxyuridine (S.4) (2.51 mmol) in a 100-mL flask. Add 20 mL anhydrous DMF under argon.
• 29Add 427 mg imidazole (2.5 eq.) and 454 mg TBDMS-Cl (1.2 eq.) and heat the mixture to 60°C in an oil bath for 2 hr under argon.
• 30Evaporate DMF using a rotary evaporator connected to a vacuum pump to give an oily residue.
• 31Add 100 mL EtOAc to the residue and wash the solution with 50 mL water, 50 mL 5% NaHCO₃ solution, and 50 mL brine, dry over Na₂SO₄, and filter.
• 32Evaporate the filtrate to dryness using a rotary evaporator to afford a crude residue.
• 33Pack a 2 × 40–cm glass column 20-cm high with silica gel in CH₂Cl₂. Apply the crude residue dissolved in a minimum of CH₂Cl₂. Elute the desired product using 0% to 2% (v/v) MeOH in CH₂Cl₂.
• 34Monitor fractions by TLC (APPENDIX 3D) using 3% MeOH in CH₂Cl₂ and combine fractions containing product. Remove solvents and dry the resulting residue using a high-performance vacuum pump to afford analytically pure nucleoside S.5 as white foam.
• 35
  Characterize the product, S.5, by ¹H NMR, ¹³C NMR, and HRMS.

  3′,5′-O-Di-t-butylsilyl-t-butyldimethylsiloxymethyl-2′-deoxyuridine (5): Yield, 1.22 g, (95%) as white foam. ¹H NMR (500 MHz) (CDCl₃) δ: 9.69 (br., 1H), 7.33 (s, 1H), 6.30 (m, 1H), 4.39–4.43 (m, 3H), 4.20 (m, 1H), 3.99 (m, 1H), 3.68 (m, 1H), 2.36 (m, 2H), 1.05 (s, 9H), 1.01 (s, 9H), 0.93 (s, 9H), 0.08 (s, 6H). ¹³C NMR (125.8 MHz) (CDCl₃) δ: 163.3, 151.2, 135.6, 116.1, 84.6, 78.9, 75.9, 68.2, 59.1, 39.6, 28.4, 28.1, 26.9, 23.6, 21.1, 19.2, −4.48, −4.49. HRMS calculated for C₂₄H₄₅N₂O₆Si, [MH⁺] 485.3047 (calcd.), 485.3041 (found).

PREPARATION OF PHOSPHORAMIDITE III

Phosphoramidite III is prepared from intermediate S.5, which is converted into the 5-hmdC analogue by first treating S.5 with POCl₃ and 1,2,4-triazole to generate a 4-triazolyl intermediate, followed by reacting with NH₄OH in dioxane to generate the amine S.6 in 65% yield in two steps. Protection of the exocyclic amine provided S.7 in 80% yield. Selective removal of the 3′,5′-silyl-protecting group of S.7 with HF in pyridine generated S.8 in 84% yield. Selective protection of the 5′-OH of S.8 with DMTr (S.9, 82%) and subsequent phosphitylation of the 3′-OH by standard procedure gave phosphoramidite III (87%) (see Fig. 4.47.3). The synthesis entails 9 steps in 18% overall yield from S.1.

Figure 4.47.3.

Synthesis of phosphoramidite III from S.5.

Materials

• 3′,5′-O-di-t-butylsilyl-t-butyldimethylsiloxymethyl-2′-deoxyuridine (S.5; see Basic Protocol 1)

• Acetonitrile (CH₃CN)

• Triethylamine (Et₃N; Sigma-Aldrich)

• Imidazole

• 1,2,4-Triazole (Chemgenes)

• Phosphorus (V) oxychloride (POCl₃; 99%, Sigma-Aldrich)

• Dichloromethane (CH₂Cl₂; anhydrous, 99.8%, Sigma-Aldrich)

• 5% NaHCO₃ solution (see recipe)

• Brine (see recipe)

• Na₂SO₄

• 1,4-Dioxane (99.8%, anhydrous, Sigma-Aldrich)

• Ammonium hydroxide (NH₄OH; 28% to 30%, Sigma-Aldrich)

• Silica gel (Sigma-Aldrich)

• Methylene chloride (CH₂Cl₂; HPLC grade, Fisher)

• Methanol (MeOH, HPLC grade, Fisher)

• Pyridine (99.8%, anhydrous, Sigma-Aldrich) Benzoyl chloride (99%, Sigma-Aldrich)

• Argon

• Toluene

  Tetrahydrofuran (THF; 99.8%, anhydrous, Sigma-Aldrich)

• Hydrogen fluoride-pyridine (HF; Sigma-Aldrich)

• DMTr-Cl (Chemgenes)

• 1-Methylimidazole (>99%, Sigma-Aldrich)

• N,N-Diisopropylethylamine (99.5%, Sigma-Aldrich)

• N,N-diisopropylamino cyanoethyl phosphoramidic-chloride (Chemgenes)

• Acetone

• 100-mL flasks

• Ice box

• Rotary evaporator connected to vacuum pump (delivering ~100 micro max vacuum), chilled by dry ice

• 2 × 40–cm glass columns

• 1 × 40–cm glass columns

• Additional reagents and equipment for TLC (APPENDIX 3D)

Prepare 3′,5′-O-di-t-butylsilyl-5-t-butyldimethylsiloxymethyl-2′-deoxycytidine (S.6)

• 1Place 1.00 g 3′,5′-O-di-t-butylsilyl-t-butyldimethylsiloxymethyl-2′-deoxyuridine (2.51 mmol, S.5) in a 100-mL flask.
• 2Add 20 mL CH₃CN, 8.56 mL Et₃N (55.3 mmol, 22 eq.), 427 mg imidazole (2.5 eq.), and 3.47 g of 1,2,4-triazole (50.5 mmol, 20.0 eq.).
• 3Cool the mixture to 0°C using an ice box. Add 0.60 mL POCl₃ (6.27 mmol, 2.5 eq.) and stir the mixture at 0°C for 0.5 hr and at room temperature for 1 hr.
• 4Add 100 mL CH₂Cl₂ and wash the mixture with 50 mL deionized water, 50 mL 5% NaHCO₃ solution, and 50 mL brine, dry over Na₂SO₄, and filter by gravity filtration (e.g., place a piece of Whatman filter paper on a conical funnel).
• 5Evaporate the filtrate to dryness using a rotary evaporator to afford a crude residue.
• 6Dissolve the residue in 10 mL 1,4-dioxane (10 mL) and cool to 0°C. Add 1 mL NH₄OH and stir the mixture at 0°C for 1 hr and then concentrate to dryness.
• 7Pack a 2 × 40–cm glass column 20-cm high with silica gel in HPLC-grade CH₂Cl₂. Apply the crude residue dissolved in a minimum of CH₂Cl₂. Elute the desired product using 0% to 4% (v/v) MeOH in CH₂Cl₂.
• 8Monitor fractions by TLC (APPENDIX 3D) using 4% MeOH in CH₂Cl₂ and combine the fractions containing product. Remove solvents using a rotary evaporator and dry the resulting residue using a high-performance vacuum pump to afford analytically pure nucleoside S.5 as white foam.
• 9
  Characterize the product, S.6, by ¹H NMR, ¹³C NMR, and HRMS.

  3′,5′-O-Di-t-butylsilyl-5-t-butyldimethylsiloxymethyl-2′-deoxycytidine (S.6): Yield, 835 mg, (65%). ¹H NMR (500 MHz) (CDCl₃) δ: 7.28 (s, 1H), 6.17 (m, 1H), 4.36–4.46 (m, 3H), 4.06 (m, 1H), 3.97 (m, 1H), 3.71 (m, 1H), 2.36 (m, 2H), 1.02 (s, 9H), 0.98 (s, 9H), 0.86 (s, 9H), 0.06 (s, 6H). ¹³C NMR (125.8 MHz) (CDCl₃) δ: 165.9, 156.3, 138.0, 106.5, 85.9, 79.0, 75.5, 68.5, 61.4, 40.3, 28.4, 28.1, 26.8, 23.6, 21.0, 19.2, −4.3. HMRS calculated for C₂₄H₄₆N₃O₅Si₂, [MH⁺] 512.2976 (calcd.), 512.2971 (found).

Prepare 3′,5′-O-di-t-butylsilyl-5-t-butyldimethylsiloxymethyl-N⁴-benzoyl-2′-deoxycytidine (S.7)

• 10Place 0.80 g 3′,5′-O-di-t-butylsilyl-5-t-butyldimethylsiloxymethyl-2′-deoxycytidine (1.56 mmol, S.6) in a 100-mL flask.
• 11Add 12 mL pyridine followed by 0.22 mL benzoyl chloride (1.87 mmol, 1.2 eq.) under argon.
• 12Stir the mixture overnight at room temperature, followed by the addition of 1 mL MeOH to quench the reaction.
• 13Evaporate the reaction mixture to dryness using a rotary evaporator followed by co-evaporating twice with 20 mL toluene to afford a crude residue.
• 14Dissolve the residue in 100 mL CH₂Cl₂, wash with 50 mL deionized water, 50 mL of 5% NaHCO₃, and 50mL brine. Dry the organic phase over Na₂SO₄ and concentrate to dryness.
• 15Pack a 2 × 40–cm glass column 20-cm high with silica gel in HPLC-grade CH₂Cl₂. Apply the crude residue dissolved in a minimum of H₂Cl₂. Elute the desired product using 0% to 2% (v/v) MeOH in CH₂Cl₂.
• 16Monitor the fractions by TLC (APPENDIX 3D) using 2% MeOH in CH₂Cl₂ and combine the fractions containing product. Remove the solvents using a rotary evaporator and dry the resulting residue using a high-performance vacuum pump to afford analytically pure nucleoside S.7 as white foam.
• 17
  Characterize the product, S.7, by ¹H NMR, ¹³C NMR, and HRMS.

  3′,5′-O-Di-t-butylsilyl-5-t-butyldimethylsiloxymethyl-N⁴-benzoyl-2′-deoxycytidine (S.7): Yield, 769 mg, (80%). ¹H NMR (500 MHz) (CDCl₃) δ: 8.24 (m, 2H), 7.58 (s, 1H), 7.54 (m, 1H), 7.45 (m, 2H), 6.29 (m, 1H), 4.70 (m, 2H), 4.48 (m, 1H), 4.22 (m, 1H), 4.04 (m, 1H), 3.85 (m, 1H), 2.41 (m, 2H), 1.08 (s, 9H), 1.06 (s, 9H), 0.96 (s, 9H), 0.18 (s, 6H). ¹³C NMR (125.8 MHz) (CD₃OD) δ: 158.4, 148.7, 137.9, 136.5, 133.6, 131.6, 130.8, 129.9, 129.1, 116.3, 85.3, 79.2, 75.7, 68.2, 59.4, 39.9, 28.4, 28.1, 27.0, 23.6, 21.1, 19.3, −4.36, −4.38. HRMS calculated for C₃₁H₅₀N₃O₆Si₂, [MH⁺] 616.3238 (calcd.), 616.3233 (found).

Prepare 5-t-butyldimethylsiloxymethyl-N⁴-benzoyl-2′-deoxycytidine (S.8)

• 18Place 720 mg 3′,5′-O-di-t-butylsilyl-5-t-butyldimethylsiloxymethyl-N⁴-benzoyl-2′-deoxycytidine (1.17 mmol, S.7) in a 100-mL flask.
• 19Add 20 mL THF followed by HF in pyridine (1.0 eq.) under argon.
• 20Stir the mixture at room temperature for 1 hr followed by addition of 4.0 g silica gel.
• 21Evaporate to dryness using a rotary evaporator to afford silica gel–mixed residue.
• 22Pack a 2 × 40–cm glass column 20-cm high with silica gel in CH₂Cl₂. Apply the crude residue and elute the desired product using 3% to 7% (v/v) MeOH in CH₂Cl₂.
• 23Monitor the fractions by TLC (APPENDIX 3D) using 7% MeOH in CH₂Cl₂ and combine the fractions containing product. Remove the solvents and dry the resulting residue using a high-performance vacuum pump to afford analytically pure nucleoside S.8 as white foam.
• 24
  Characterize the product, S.8, by ¹H NMR, ¹³C NMR, and HRMS.

  5-t-Butyldimethylsiloxymethyl-N⁴-benzoyl-2′-deoxycytidine (S.8): Yield, 476 mg, (84%). ¹H NMR (500 MHz) (Aceton-d₆) δ: 8.26 (m, 2H), 8.15 (s, 1H), 7.56 (m, 1H), 7.47 (m, 2H), 6.34 (m, 1H), 4.73 (s, 2H), 4.52 (m, 1H), 4.03 (m, 1H), 3.80 (m, 1H), 2.39 (m, 1H), 2.30 (m, 1H), 0.97 (s, 9H), 0.18 (s, 6H). ¹³C NMR (125.8 MHz) (Aceton-d₆) δ: 159.7, 148.6, 139.6, 138.2, 133.3, 130.6, 129.0, 114.9, 89.1, 87.0, 72.3, 63.0, 59.4, 41.47, 26.5, 19.1, −5.0. HRMS calculated for C₂₃H₃₄N₃O₆Si, [MH⁺] 476.2217 (calcd.), 476.2211 (found).

Prepare 5′-O-(4,4′-dimethoxytrityl)-5-t-butyldimethylsiloxymethyl-N⁴-benzoyl-2′-deoxycytidine (S.9)

• 25Place 412 mg 5-t-butyldimethylsiloxymethyl-N⁴-benzoyl-2′-deoxycytidine (0.87 mmol, S.8) in a 100-mL flask.
• 26Add 12 mL pyridine followed by 352 mg DMTr-Cl (1.04 mmol, 1.2 eq.) under argon.
• 27Stir the mixture overnight at room temperature, followed by addition of 1 mL MeOH to quench the reaction.
• 28Evaporate the solvent to dryness using a rotary evaporator followed by co-evaporating twice with 20 mL toluene to afford a residue.
• 29Dissolve the residue in 100 mL CH₂Cl₂, wash with 50 mL deionized water, 50 mL of 5% NaHCO₃, and 50 mL brine. Dry the organic phase over Na₂SO₄ and concentrate to dryness.
• 30Pack a 2 × 40–cm glass column 20-cm high with silica gel in CH₂Cl₂. Apply the crude residue and elute the desired product using 0% to 3% (v/v) MeOH in CH₂Cl₂.
• 31Monitor the fractions by TLC (APPENDIX 3D) using 3% MeOH in CH₂Cl₂ and combine the fractions containing product. Remove the solvents using a rotary evaporator and dry the resulting residue using a high-performance vacuum pump to afford analytically pure nucleoside S.9 as white foam.
• 32
  Characterize the product, S.9, by ¹H NMR, ¹³C NMR, and HRMS.

  5′-O-(4,4′-Dimethoxytrityl)-5-t-butyldimethylsiloxymethyl-N⁴-benzoyl-2′-deoxy-cytidine (S.9): Yield, 553 mg, (82%). ¹H NMR (500 MHz) (CD₃CN) δ: 8.24 (d, J=8.0 Hz, 2H), 7.71 (s, 1H), 7.24-7.58 (m, 12H), 6.87 (m, 4H), 6.22 (m, 1H), 4.57 (m, 1H), 4.40 (m, 1H), 4.28 (m, 1H), 3.98 (m, 1H), 3.76 (s, 6H), 3.34 (m, 1H), 3.29 (m, 1H), 3.22 (m, 1H), 2.39 (m, 1H), 2.18 (m, 1H), 0.83 (s, 9H), 0.05 (s, 3H), 0.04 (s, 3H). ¹³C NMR (125.8 MHz) (CD₃CN) δ: 159.7, 146.0, 139.3, 137.8, 136.9, 136.8, 133.5, 131.0, 130.9, 130.4, 129.2, 129.0, 128.9, 127.9, 114.1, 87.3, 86.8, 86.6, 71.9, 64.8, 59.5, 55.9, 40.8, 26.3, 18.9, −5.1. HRMS calculated for C₄₄H₅₂N₃O₈Si, [MH⁺] 778.3524 (calcd.), 778.3525 (found).

Prepare 5′-O-(4,4′-dimethoxytrityl)-5-t-butyldimethylsiloxymethyl-N⁴-benzoyl-2′-deoxycytidine 3′-O-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite (III)

• 33Place 120 mg 5′-O-(4,4′-dimethoxytrityl)-5-t-butyldimethylsiloxymethyl-N⁴-benzoyl-2′-deoxycytidine (0.87 mmol, S.9) in a 100-mL flask.
• 34Add 8 mL anhydrous CH₂Cl₂, 3.40 mg (41.5 µmol) of 1-methylimidazole, and 0.2 mL N,N-diisopropylethylamine under argon, followed by the addition of 71.7 mg (0.30 mmol) N,N-diisopropylamino cyanoethyl phosphoramidic-chloride.
• 35Stir the mixture at room temperature for 0.5 hr, followed by the addition of 80 mL CH₂Cl₂.
• 36Wash the solution with 40 mL deionized water, 40 mL of 5% NaHCO₃, and 40 mL brine. Dry the organic phase over Na₂SO₄ and concentrate to dryness.
• 37Pack a 1 × 40–cm glass column 20-cm high with silica gel in CH₂Cl₂. Apply the crude residue and elute the desired product using 6% to 9% acetone in CH₂Cl₂ containing 0.2% Et₃N.
• 38Monitor the fractions by TLC (APPENDIX 3D) using 9% acetone in CH₂Cl₂ and combine the fractions containing product. Remove the solvents using a rotary evaporator and dry the resulting residue using a high-performance vacuum pump to afford analytically pure nucleoside S.9 as white foam.
• 39
  Characterize the product, III, by ¹H NMR, ³¹P NMR, and HRMS.

  5′-O-(4,4′-Dimethoxytrityl)-5-t-butyldimethylsiloxymethyl-N⁴-benzoyl-2′-deoxycytidine (III): Yield, 131 mg, (87%). ¹H NMR (500 MHz) (CD₃CN) δ: 8.30 (m, 2H), 7.31–7.72 (m, 13H), 6.87 (m, 4H), 6.18–6.28 (m, 1H), 4.62 (m, 1H), 4.40–4.50 (m, 2H), 4.15 (m, 1H), 3.76 (m, 6H), 3.65 (m, 4H), 3.32 (m, 2H), 2.65 (m, 2H), 2.54 (m, 2H), 2.28 (m, 1H), 2.16 (m, 1H), 1.17 (m, 9H), 1.05 (m, 3H), 0.84 (s, 9H), 0.04 (s, 6H). ³¹P NMR (202.5 MHz) (CD₃CN) 148.44 ppm. HRMS calcd. for C₅₃H₆₉N₅O₉PSi, [MH]⁺ 978.4602 (calcd.), 978.4611 (found).

PREPARATION OF PHOSPHORAMIDITE IV

So far, all the syntheses of phosphoramidite building blocks I, II, and III required a multiple-step transformation of converting 5-hmU derivatives to the corresponding 5-hmC analogues in two steps and an additional step to protect the resulting exocyclic amine with Bz. To simplify the synthesis, we chose to synthesize another version of phosphoramidite IV that bears 4-triazoylide instead of 4-BzNH. Since 4-triazolide can be converted simultaneously into 4-NH₂ during post-synthetic NH4OH treatment, two steps of the reaction could therefore be spared. The same strategy has been successfully used for synthesizing nucleotides containing 4-NH₂, 4-MeO, 4-EtO, 4-Me₂N-NH, and 4-SH analogues (Xu et al., 1992). Besides allowing for the incorporation of 5-hmdC into DNA, phosphoramidite IV may also find applications in synthesizing DNA containing 4-substituted 5-hydroxymethylpyrimidine derivatives.

The synthesis of phosphoramidite IV starts from intermediate S.5 (Fig. 4.47.4). Selective removal of the 3′,5′-silyl protecting group of S.5 with HF-pyridine generates S.10 in 88% yield. Selective protection of the 5′-OH of S.10 with DMTr (S.11, 85%) and subsequent phosphitylation of the 3′-OH by standard procedure gives phosphoramidite S.12 (92%). Phosphoramidite S.12 is quantitatively converted to phosphoramidite IV although it has a silylated hydroxymethyl group in the adjacent 5-position (Tardy-Planechaud et al., 1997). Phosphoramidite IV is also stable during silica gel chromatography and is isolated in 81% yield. The synthesis entails 8 steps and the overall yield from S.1 reaches 32%.

Figure 4.47.4.

Synthesis of phosphoramidite IV from S.5.

Materials

• 3′,5′-O-di-t-butylsilyl-t-butyldimethylsiloxymethyl-2′-deoxyuridine (S.5; see Basic Protocol 1)

• Tetrahydrofuran (THF; 99.8%, anhydrous, Sigma-Aldrich)

• Hydrogen fluoride-pyridine (HF; Sigma-Aldrich)

• Silica gel (Sigma-Aldrich)

• Methylene chloride (CH₂Cl₂; HPLC grade, Fisher)

• Methanol (MeOH; HPLC grade, Fisher)

• Pyridine (99.8%, anhydrous, Sigma-Aldrich)

• DMTr-Cl (Chemgenes)

• Argon

• Toluene

• 5% NaHCO₃ solution (see recipe)

• Brine (see recipe)

• Na₂SO₄

• Dichloromethane (CH₂Cl₂; anhydrous, 99.8%, Sigma-Aldrich)

• 1-Methylimidazole (>99%, Sigma-Aldrich)

• N,N-Diisopropylethylamine (99.5%, Sigma-Aldrich)

• N,N-diisopropylamino cyanoethyl phosphoramidic-chloride (Chemgenes)

• Acetone

• Triethylamine (Et₃N; Sigma-Aldrich)

• Acetonitrile (CH₃CN; anhydrous, 99.8%, Sigma-Aldrich)

• 1,2,4-Triazole (Chemgenes)

• Phosphorus (V) oxychloride (POCl₃; 99%, Sigma-Aldrich)

• 100-mL flasks

• Rotary evaporator connected to vacuum pump (delivering ~100 micro max vacuum), chilled by dry ice

• 2 × 40–cm glass columns

• 1 × 40–cm glass columns

  Additional reagents and equipment for TLC (APPENDIX 3D)

Prepare synthesis 5-t-butyldimethylsiloxymethyl-2′-deoxyuridine (S.10)

• 1Place 440 mg 3′,5′-O-di-t-butylsilyl-t-butyldimethylsiloxymethyl-2′-deoxyuridine (2.51 mmol, S.5) in a 100-mL flask.
• 2Add 20 mL THF followed by HF-pyridine (1.0 eq.) under argon.
• 3Stir the mixture at room temperature for 1 hr followed by addition of 3.0 g silica gel.
• 4Evaporate to dryness using a rotary evaporator to afford silica gel–mixed residue.
• 5Pack a 2 × 40–cm glass columns 20-cm high with silica gel in CH₂Cl₂. Apply the crude residue and elute the desired product using 0% to 7% (v/v) MeOH in CH₂Cl₂.
• 6Monitor fractions by TLC (APPENDIX 3D) using 7% MeOH in CH₂Cl₂ and combine the fractions containing product. Remove and dry the resulting residue using a high-performance vacuum pump to afford analytically pure nucleoside S.10 as white foam.
• 7
  Characterize the product, S.10, by ¹H NMR, ¹³C NMR, and HRMS.

  5-t-Butyldimethylsiloxymethyl-2′-deoxyuridine (S.10): Yield, 281 mg, (88%). ¹H NMR (500 MHz) (Aceton-d₆) δ: 10.07 (br., 1H), 7.83 (s, 1H), 6.34 (m, 1H), 4.50 (s, 1H), 4.42 (s, 3H), 4.12 (m, 1H), 3.95 (m, 1H), 3.75 (m, 2H), 2.26 (m, 2H), 0.92 (s, 9H), 0.12 (s, 6H). ¹³C NMR (125.8 MHz) (Aceton-d₆) δ: 163.0, 151.3, 137.7, 114.7, 88.7, 85.9, 72.5, 63.2, 59.2, 41.0, 26.4, 19.0, −5.1. HRMS calculated for C₁₆H₂₉N₂O₆Si, [MH⁺] 373.1795 (calcd.), 373.1789 (found).

Prepare 5′-O-(4,4′-dimethoxytrityl)-5-t-butyldimethylsiloxymethyl-2′-deoxyuridine (S.11)

• 8Place 260 mg 5-t-butyldimethylsiloxymethyl-2′-deoxyuridine (0.70 mmol, S.10) in a 100-mL flask.
• 9Add 8 mL pyridine followed by 284 mg DMTr-Cl (0.84 mmol, 1.2 eq.) under argon.
• 10Stir the mixture overnight at room temperature followed by addition of 1 mL MeOH to quench the reaction.
• 11Evaporate the solvent to dryness using a rotary evaporator followed by co-evaporating twice with 20 mL toluene to afford a residue.
• 12Dissolve the residue in 100 mL CH₂Cl₂, wash with 50 mL water, 50 mL of 5% NaHCO₃, and 50 mL brine. Dry the organic phase over Na₂SO₄ and concentrate to dryness.
• 13Pack a 2 × 40–cm glass column 20-cm high with silica gel in CH₂Cl₂. Apply the crude residue and elute the desired product using 0% to 3% (v/v) MeOH in CH₂Cl₂.
• 14Monitor fractions by TLC (APPENDIX 3D) using 7% MeOH in CH₂Cl₂ and combine the fractions containing product. Remove solvents using a rotary evaporator and dry the resulting residue using a high-performance vacuum pump to afford analytically pure nucleoside S.9 as white foam.
• 15
  Characterize the product, S.11, by ¹H NMR, ¹³C NMR, and HRMS.

  5′-O-(4,4′-Dimethoxytrityl)-5-t-butyldimethylsiloxymethyl-2′-deoxyuridine (S.11): Yield, 400 mg, (85%). ¹H NMR (500 MHz) (CD₃CN) δ: 9.34 (br., 1H), 7.46 (m, 3H), 7.24–7.35 (m, 7H), 6.87 (m, 4H), 6.23 (m, 1H), 4.29 (m, 2H), 4.12 (m, 1H), 3.95 (m, 1H), 3.77 (s, 6H), 3.29 (m, 1H), 3.20 (m, 1H), 2.30 (m, 1H), 2.19 (m, 1H), 0.84 (s, 9H), 0.02 (s, 3H), 0.01 (s, 3H). ¹³C NMR (125.8 MHz) (CD₃CN) δ: 163.3, 159.7, 151.3, 146.0, 137.6, 136.8, 131.0, 128.98, 128.90, 127.89, 118.3, 115.0, 114.1, 87.3, 86.0, 85.4, 72.1, 65.0, 59.0, 55.9, 40.5, 26.3, 18.9, 7.5. HRMS calculated for C₃₇H₄₇N₂O₈Si, [MH⁺] 675.3102 (calcd.), 675.3096 (found).

Prepare 5′-O-(4,4′-dimethoxytrityl)-5-t-butyldimethylsiloxymethyl-2′-deoxyuridine 3′-O-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite (S.12)

• 16Place 120 mg 5′-O-(4,4′-dimethoxytrityl)-5-t-butyldimethylsiloxymethyl-2′-deoxyuridine (0.18 mmol, S.11) in a 100-mL flask.
• 17Add 8 mL anhydrous CH₂Cl₂, 3.40 mg of 1-methylimidazole (41.5 µmol), and 0.2 mL N,N-diisopropylethylamine under argon, followed by addition of 60 µL N,N-diisopropylamino cyanoethyl phosphoramidic-chloride (0.27 mmol).
• 18Stir the mixture at room temperature for 0.5 hr followed by addition of 80 mL CH₂Cl₂.
• 19Wash the solution with 40 mL water, 40 mL of 5% NaHCO₃, and 40 mL brine. Dry the organic phase over Na₂SO₄ and concentrate to dryness.
• 20Pack a 1 × 40–cm glass column 20-cm high with silica gel in CH₂Cl₂. Apply the crude residue and elute the desired product using 6% to 9% acetone in CH₂Cl₂ containing 0.2% Et₃N.
• 21Monitor the fractions by TLC (APPENDIX 3D) using 9% acetone in CH₂Cl₂ and combine the fractions containing product. Remove solvents using a rotary evaporator and dry the resulting residue using a high-performance vacuum pump to afford analytically pure nucleoside S.12 as white foam.
• 22
  Characterize the product, S.12, by ¹H NMR, ³¹P NMR, and HRMS.

  5′-O-(4,4′-Dimethoxytrityl)-5-t-butyldimethylsiloxymethyl-2′-deoxyuridine 3′-O-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite (S.12): Yield, 143 mg (92%). ¹H NMR (500 MHz) (CD₃CN) δ: 9.04 (br., 1H), 7.03-7.46 (m, 10H), 6.89 (m, 4H), 6.23 (m, 1H), 4.50 (m, 1H), 4.28 (m, 1H), 4.08 (m, 2H), 3.78 (s, 3H), 3.77 (s, 3H), 3.62 (m, 4H), 3.28 (m, 2H), 2.63 (m, 1H), 2.53 (m, 1H), 2.44 (m, 1H), 2.26 (m, 1H), 1.17 (m, 9H), 1.05 (d, 3H), 0.82 (s, 9H), 0.02 (s, 3H), 0.00 (s, 3H). ³¹P NMR (202.5 MHz) (CD₃CN) 148.34, 148.27 ppm. HRMS calculated for C₄₆H₆₆N₄O₉PSi, [MH⁺] 877.4337 (calcd.), 877.4331 (found).

Prepare 5′-O-(4,4′-Dimethoxytrityl)-5-t-butyldimethylsiloxymethyl-4-triazolothymidine 3′-O-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite (IV)

• 23Place 110 mg 5′-O-(4,4′-dimethoxytrityl)-5-t-butyldimethylsiloxymethyl-2′-deoxyuridine 3′-O-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite (S.12) in a 100-mL flask.
• 24Add 5 mL CH₃CN, 0.43 mL Et₃N, and 174 mg 1,2,4-triazole. Cool the mixture to 0°C.
• 25Add 30 µL POCl₃. Stir the mixture at 0°C for 0.5 hr and at room temperature for 1 hr. Add another portion of POCl₃ (16 µL) and stir the mixture at room temperature for 5 hr.
• 26Add 100 mL CH₂Cl₂ and wash the mixture with 50 mL water, 50 mL of 5% NaHCO₃, and 50 mL brine. Dry over Na₂SO₄ and concentrate to dryness.
• 27Pack a 1 × 40–cm glass column 20-cm high with silica gel in CH₂Cl₂. Apply the crude residue dissolved in a minimum of CH₂Cl₂. Elute the desired product using 6% to 10% acetone in CH₂Cl₂ containing 0.2% Et₃N.
• 28Monitor the fractions by TLC (APPENDIX 3D) using 10% acetone in CH₂Cl₂ and combine the fractions containing product. Remove the solvents using a rotary evaporator and dry the resulting residue using a high-performance vacuum pump to afford analytically pure nucleoside IV as white foam.
• 29
  Characterize the product, IV, by ¹H NMR, ¹³C NMR, ³¹P NMR, and HRMS.

  5′-O-(4,4′-Dimethoxytrityl)-5-t-butyldimethylsiloxymethyl-4-triazolothymidine 3′-O-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite (IV): Yield, 94.5 mg (81%). ¹H NMR (500 MHz) (CD₃CN) δ: 9.18 (s, 1H), 8.50 (s, 0.5H), 8.38 (s, 0.5H), 8.15 (s, 1H), 7.24–7.42 (m, 9H), 6.87 (m, 4H), 6.18 (m, 1H), 4.92 (m, 1H), 4.55 (m, 2H), 4.22 (m, 1H), 3.75 (s, 6H), 3.73 (m, 4H), 3.40 (m, 2H), 2.66 (m, 1H), 2.56 (m, 1H), 2.25 (m, 2H), 1.17 (m, 9H), 1.07 (d, 3H), 0.79 (s, 9H), −0.04, −0.03 (s, 3H), 0.00 (s, 3H). ¹³C NMR (125.8 MHz) (CD₃CN) δ: 159.7, 154.5, 154.5, 147.1, 147.0, 145.6, 136.6, 131.0, 131.0, 129.0, 128.9, 127.98, 127.95, 114.2, 89.9, 88.8, 89.0, 88.1, 88.0, 74.6, 74.4, 64.2, 60.9, 60.9, 59.5, 59.3, 55.9, 55.9, 44.1, 44.02, 44.00, 40.1, 40.1, 26.3, 26.2,, 25.0, 24.9, 24.8, 21.0, 18.6, −5.15, −5.16. ³¹P NMR (202.5 MHz) (CD₃CN) 148.31, 148.24 ppm. HMRS calculated for C₄₈H₆₅N₇O₈PSi, [MH⁺] 926.4402 (calcd.), 926.4396 (found).

SOLID-PHASE SYNTHESIS OF DNA USING PHOSPHORAMIDITES III AND IV AND SUBSEQUENT DEPROTECTION

To optimize the coupling and deprotection conditions, we incorporated phosphoramidites III and IV into a short model sequence 5′-TCXGA (X = 5-hmC) using ultramild reagents to give oligodeoxynucleotides ODN1 and ODN2, respectively. When using the standard protocol to do 1-µmol solid-phase synthesis, the DMTr cation release showed that the coupling efficiency of phosphoramidite III or IV was as efficient as that of wild-type phosphoramidites. Following the deprotection of ODN1 and ODN2, we analyzed the coupling by running C18 reversed-phase HPLC with a gradient of 0% to 18% acetonitrile in 0.1 M TEAA within 30 min. The HPLC analysis showed that more dimers were formed (due to the failed coupling of phosphoramidite III or IV) than 3mer and 4mer (due to the failed coupling of wild-type phosphoramidites G or A), suggesting that the coupling yields of phosphoramidite III and IV are not as good as wild-type phosphoramidites (phosphoramidite IV coupled better than III). To improve their coupling yields, we modified the coupling program so that phosphoramidite III or IV underwent double-coupling. For comparison, we also incorporated phosphoramidite II from Glen Research into the same sequence under the same conditions to give ODN3.

Following oligosynthesis, we first treated ODN2 with concentrated NH₄OH at room temperature overnight. Reversed-phase HPLC analysis (Fig. 4.47.5A) showed that, besides DNA with a TBDMS-protecting group (peak c), over 20% of DNA without TBDMS group was also produced (peak b). It is interesting to note that the 5-CH₂O-TBDMS group is much more labile to the NH₄OH treatment than the regular 2′-O-TBDMS-protecting group in RNA synthesis, which can survive the overnight treatment at 55°C. This observation is consistent with the enhanced reactivity of the 5-CH₂OH in S.2 as indicated by selective protection of 5-CH₂OH by TBDMS or Ac against the 5′-hydroxyl (Conte et al., 1992; Sowers and Beardsley, 1993). The liability of the 5-CH₂O-TBDMS ether to the NH₄OH treatment suggests that it is possible to fully remove it from the oligo at elevated temperature so that the extra fluoride treatment might be spared. Indeed, when we treated ODN2 with NH₄OH at 65°C for 16 hr, HPLC analysis indicated that TBDMS was almost fully removed (Fig. 4.47.5B). The same treatment of ODN1 also gave the same major desired product. In contrast, ODN3 only gave about 56% of the desired product, while another 44% with the 5-cyanoethyl protecting group still hung on (Fig. 4.47.5C), suggesting that the TBDMS is a superior protecting group to the cyanoethyl.

Figure 4.47.5.

Reversed-phase HPLC analysis using C18 column with a gradient of 0% to 18% CH₃CN in 0.1 M TEAA from 6 to 36 min. (A) ODN2 was deprotected by treatment of NH₄OH overnight at room temperature. Peak a is a failed sequence dimer, 5′-CT ([MH]⁺ = 531); peak b is the fully deprotected 5mer 5′-GAXCT ([MH]⁺ = 1493); peak c is the 5mer with TBDMS-protecting group on 5hmC ([MH]⁺ = 1607). (B) ODN2 was deprotected by treatment of NH₄OH overnight at 65°C. (C) ODN3 was deprotected by treatment of NH₄OH overnight at 65°C; peak d is the 5mer with cyanoethyl-protecting group on 5hmC ([MH]⁺ = 1546). (D) ODN2 was deprotected by treatment of 0.5 M NH₄F in MeOH overnight, followed by NH₄OH treatment at room temperature for 2 hr.

The above one-step deprotection procedure is very convenient for the synthesis of DNA containing 5-hmC. For DNA containing additional labeling modifications that requires milder deprotection, a prior treatment with fluoride to remove the TBDMS group may allow for the use of ultramild deprotection of the synthetic DNA. Thus, after treating the solid resin bearing newly synthesized ODN2 with NH₄F (0.5 M in MeOH) overnight at room temperature to remove the TBDMS group and rinsing with MeOH to get rid of excess NH₄F, we applied the ultramild deprotection condition (NH₄OH treatment at room temperature for 2 hr) to the resin. HPLC analysis showed that all the protecting groups were cleanly removed and the desired product (peak b) was produced as the major product (Fig. 4.47.5D). The same treatment of ODN1 also gave fully deprotected oligo. This is surprising because 4-benzoyl protecting group of cytidine is usually not compatible with ultramild deprotection conditions; the 5-CH₂OH probably facilitates the removal of benzoyl group in the presence of NH₄OH.

REAGENTS AND SOLUTIONS

Use deionized, distilled water in all recipes and protocol steps. For common stock solutions, see APPENDIX 2A; for suppliers, see SUPPLIERS APPENDIX.

Brine

Add 200 g NaCl to a 1-L bottle and add deionized water until the volume is 1 L. Shake the bottle to dissolve the solid as much as possible. Use the supernatant as the brine.

NaHCO₃ solution, 5%

Add 50 g NaHCO₃ into a 1-L bottle and add deionized water until the volume is 1 L. Shake the bottle to dissolve the solid to give 5% NaHCO₃ solution.

COMMENTARY

Background Information

5-Methylcytosine (5-mC) is an important DNA modification found in eukaryotes and is referred to as the fifth base besides dA, dC, dG, and dT. It constitutes ~2% to 8% of the total cytosine in human genomic DNA, and impacts a broad range of biological functions including gene expression, maintenance of genome integrity, parental imprinting, X-chromosome inactivation, regulation of development, aging, and cancer (Goll and Bestor, 2005; Klose and Bird, 2006; Reik, 2007; Weber and Schubeler, 2007; Gal-Yam et al., 2008). In 2009, 5-hydroxymethylcytosine (5-hmC), an oxidized form of 5-mC, was discovered in substantial amounts in the mammalian genome of certain cell types as the “sixth” base (Kriaucionis and Heintz, 2009; Tahiliani et al., 2009). Recent studies have shown that 5-hmC is a widespread DNA modification in brain tissues and stem cells, and its abundance is dependent on tissue type (Münzel et al., 2010a; Szwagierczak et al., 2010). We have recently developed an efficient method to specifically label and pull down 5-hmC-containing DNA fragments for subsequent deep sequencing to reveal the genome-wide distribution of 5-hmC, which in mouse cerebellum appears to be enriched in a gene expression level-dependent manner (Song et al., 2011; Moran-Crusio et al., 2011). In addition, it has been hypothesized that the TET protein-catalyzed conversion of 5-mC DNA to 5-hmC may represent the first step of multiple-step reactions for DNA demethylation (Wu and Zhang, 2010; Globisch et al., 2010; Dai and He, 2011). Further oxidation of 5-hmC in DNA may result in the formation of 5-formylcytosine (5-fC) DNA. It is possible that 5-fC is a transient intermediate, which can be quickly converted to 5-carboxylcytosine (5-caC) in vivo and cytosine by further decarboxylation (Radzicka and Wolfenden, 1995). Indeed, using the 5-hmC-containing DNA oligos prepared by the method in this unit together with the corresponding 5-fC and 5-caC-containing oligos we synthesized (Dai et al., 2011), we and our collaborators recently found strong evidences that 5-hmC can be further oxidized to 5-fC and 5caC by all the three Tet proteins. (He et al., 2011; Ito et al., 2011).

Compound characterization

Chemical characterization data are provided for all new compounds. NMR spectra were recorded on a Bruker 500 or Bruker 400 MHz NMR spectrometer. The ¹H, ¹³C, and, ³¹P NMR chemical shifts are described as δ values in ppm relative to Me₄Si (for ¹H and ¹³C) and 85% H₃PO₄ (for ³¹P). High-resolution mass spectra (HRMS) were obtained from Notre Dame Mass Spectrometry & Proteomics Facility. Maldi-MS was recorded on a Voyager Biospectrometry Workstation with THAP as matrix.

Relevant work

While the current work was going on, another 5-hmC phosphoramidite using a carbamate to protect the amino and hydroxyl groups simultaneously was reported (Münzel et al. 2010b). The synthesis derives from 5-iododeoxycytidine and entails seven steps of reaction and gave the phosphoramidite in 7.5% overall yield. Although a significant advance compared to the commercial available phosphoramidite, the synthesis is still not compatible with ultramild deprotection conditions. After this work, we found that DNA oligos containing 5-fC modification, which were prepared by ultramild DNA synthesis (Dai and He, 2011), could be quantitatively converted to the corresponding 5-hmC oligos by the reduction of NaBH₄. It offered an alternative method for ultramild synthesis of DNA oligos containing 5-hmC modification.

Critical Parameters and Troubleshooting

The purity of all solvents and reagents is critical to obtaining high yields and pure compounds. Where specified, solvents must be anhydrous. Running reactions under an inert atmosphere, such as argon, is good laboratory practice. The syntheses described above require understanding of and experience with common organic synthetic procedures. It is important that intermediates be pure for subsequent steps.

The transformation from S.2 to S.3 required 50 psi of CO in the reactor. We also tried the reaction in the 1 atm atmosphere of CO, but found there was a significant amount of 3′,5′-O-di-t-butylsilyl-5-formyl-2′-deoxyuridine formed as a byproduct. The quality of tributyltin hydride is also important. It should be stored at 0°C. It may lose its reducing ability after storing at room temperature for a long time. Addition of tributyltin hydride too quickly may also result in the formation of the byproduct.

The transformation from S.12 to IV is tricky. The excess amount of reagents, such as Et₃N and 1,2,4-triazole, is important, but adding too much POCl₃ may result in the decomposition of the resulting phosphoramidite IV. Use TLC to monitor the reaction until starting material S.12 is completely consumed. Otherwise, phosphoramidite IV may be contaminated with S.12, which will result in the contamination of 5-hmU in the synthesized DNA oligo.

Anticipated Results

An experienced synthetic chemist should be able to obtain yields comparable to those reported (the overall yield for phosphoramidite III and IV is 18% and 32%, respectively), if the procedures are followed carefully. Less skilled researchers may require several attempts to master the reactions, but should eventually succeed, provided that the procedures are followed exactly.

Time Considerations

Each of the intermediates described in this unit can be prepared within 1 or 2 days. Synthesis of the phosphoramidite IV is by far the shortest (8 days) compared to that of III (10 days). We also use phosphoramidites III and IV for longer ODN synthesis (e.g., 32mer) and we did not encounter any problems. However, we prefer to use phosphoramidite IV since it is easier to prepare.

Solid-phase synthesis (1-µmol scale), deprotection, and purification of three to four 5-hmC ODN of 20 to 32 nt by HPLC can typically be achieved within 5 to 7 days.